Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease

Summary Background Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. Methods The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. Findings Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. Interpretation Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. Funding This study was funded by the 10.13039/100010269Wellcome Trust (211113/A/18/Z).


Introduction
CD4 + CD25 + Foxp3 + Regulatory T cells (Treg) are known to modulate inflammatory responses by suppressing effector T cells, inhibiting neutrophils and proinflammatory macrophages, and promoting the activities of anti-inflammatory macrophages and monocyte subsets. 1 Tregs are of paramount importance to maintain immune homeostasis, as their dysfunction leads to the development of systemic inflammation, autoimmunity, and immunopathology. 2 Under steady state, Tregs maintain a stable immunosuppressive phenotype.However, in certain inflammatory environments (e.g.IL-6, 3,4 TNFα 5,6 ) Treg function can be compromised in number and/or function, [7][8][9] exacerbating the underlying disease.Moreover, our own previous work indicates that reactive oxygen species (ROS), inherently associated to

Research in context
Evidence before this study Chronic liver damage driven by both inflammation and oxidative stress often leads to cirrhosis (LC).Without treatment, patients with LC inexorably progress to clinical decompensations and death.The most advanced forms of LC are characterized by the development of systemic inflammation and innate immune deficits, known as cirrhosisassociated immune dysfunction (CAID), which is believed to contribute to multi-organ failure and death.Regulatory T cells (Tregs) are a subset of immune cells that are critical to curb excessive immune activation, to maintain immune and tissue homeostasis, and to prevent autoimmunity.The role of Tregs in the natural history of chronic liver disease as well as their involvement in the immunological abnormalities associated with LC has not been investigated in humans.

Added value of this study
Our study has revealed a numerical and functional deficit of circulating Tregs in patients with LC, which correlates with liver disease severity and resembles what has been described in many autoimmune diseases.In contrast to most published studies describing CAID, our analyses were conducted on specimens collected from stable LC patients without acute decompensations, thus avoiding the risks of clinical confounders (e.g.sepsis, extra-hepatic organ failure, medications).From a mechanistic standpoint, we have identified a dysregulated intracellular redox homeostasis as a key element for the Treg abnormalities observed in LC, which is mainly driven by the impairment of the anti-oxidant Nrf2 signalling pathway and a resultant loss of mitochondrial function and integrity.Furthermore, our findings demonstrate the role of the cytoprotective enzyme HO-1, a downstream target of Nrf2 signalling, in promoting Treg survival under oxidative stress conditions.
Implications of all the available evidence Our finding that in LC patients at early stages of their disease circulating Tregs are already dysfunctional fills a gap in our understanding of the immunopathogenesis of the disease.What our results suggest is that clinically stable LC are already predisposed to develop the dysregulated inflammatory responses that constitute the hallmark of decompensated LC and that contribute to poor prognosis.This implies that it may be possible to prevent clinical decompensations and LC progression, thus improving patient survival, by therapeutically restoring Treg function.In addition, from our results demonstrating the importance of redox homeostasis in the maintenance of Treg viability and function, we infer that similar mechanisms could be at play in other pathological conditions mediated by oxidative damage that also influence Treg function (e.g.ageing, cancer, cardiovascular diseases).The availability of multiple druggable targets in the redox homeostasis machinery provide potential opportunities to correct Treg dysfunction in these settings.A note of caution is however due, given the need to conduct further studies to identify the specific mediators responsible for Treg dysfunction in LC, and to establish links between the magnitude of the dysfunction and clinical outcomes.inflammation as a result of immune cell activation, 10,11 can also induce Treg dysfunction. 12,13Thus, we described a link between the expression of the ROSgenerating NADPH oxidase-2 (Nox2) enzyme and Treg phenotype and function, with Nox2-deficient Tregs exhibiting higher suppressive capacity than wild type Tregs, both in vitro and in vivo. 12,13mong the various signalling pathways modulating intracellular redox balance, the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as a primary cellular defence against the cytotoxic effects of oxidative stress. 14In response to inflammation and ROS, Nrf2 translocates to the nucleus 14,15 and upregulates a variety of antioxidant, anti-apoptotic and antiinflammatory enzymes, among others hemeoxygenase-1 (HO-1). 14,16,17HO-1 exerts powerful cytoprotective effects in various chronic inflammatory diseases 18 through the degradation of heme, a pro-oxidant molecule whose bioavailability is increased as a result of tissue injury, into its by-products ferrous iron, carbon monoxide and biliverdin. 19,202][23] HO-1 is constitutively expressed by human Tregs 24 and is involved in FOXP3-mediated suppression, 25 in the production of IL-10 26 and in the protection of Tregs from cellular damage. 27Taken together with our previous findings, 13 the evidence outlined above raise the possibility, yet to be investigated, of the Nrf2/HO-1 and Nox2 balance being a key determinant of Treg function in clinical settings characterized by both excessive inflammation and oxidative stress.
Liver cirrhosis (LC) constitutes the end stage of most chronic inflammatory liver diseases.Left unchecked, LC evolves towards the development of acute clinical decompensations, multi-organ failures, and eventually death. 28As shown in a multiplicity of studies, LC is associated with excessive oxidative stress [29][30][31][32] and leads to a severely dysregulated systemic immune system, characterised by both immunodeficiency and increased inflammation. 33,34This phenomenon is believed to be responsible for the propensity of LC patients to develop extra-hepatic organ failures, as well as for their increased susceptibility to infections and their high mortality as a result of sepsis.
In the current study, we sought to investigate the extent to which intracellular redox balance influences human Treg homeostasis and function, using LC as paradigmatic disease model of oxidative stress and systemic inflammation.To this end, we conducted an exhaustive phenotypic and functional characterisation of circulating Tregs from clinically stable LC patients.Furthermore, we performed mechanistic investigations to elucidate whether the expression and activity of anti-oxidant and pro-oxidant enzymes alters Treg function using both clinical specimens and a relevant animal model.This study sheds new light on the impact of excessive oxidative stress on Treg function in humans.We propose that modulation of Treg redox homeostasis may represent a strategy to counteract the dysregulated systemic inflammation driving clinical complications in LC patients.

Study participants
A total of 108 patients with stable LC were prospectively recruited from the liver outpatient clinic at King's College Hospital from September 2015 until June 2023.From these patients, 103 were diagnosed with alcoholrelated cirrhosis (ARC), abstinent for at least 6 months, and 5 patients with non-alcoholic steatohepatitis (NASH).In parallel 71 healthy controls (HC) were recruited and used as comparators (Table 1).
To eliminate confounders known to influence lymphocyte function, patients with infections in the preceding three months, acute-on-chronic liver failure, previous transplantation, autoimmune diseases, hepatotropic viral disease, cholestatic liver disease, haemochromatosis, hepatocellular carcinoma, and/or those on iron supplementation or antibiotics (including rifaximin) were excluded.

Ethics and study approval
The study was conducted in line with the Declaration of Helsinki and approved by the Institutional Review Board of Guy's hospital (reference 09/H0707/86).Informed consent was obtained from all subjects enrolled into the study.All blood samples were handled and disposed of in accordance with the UK Human Tissue Act 2008.The animal study was performed in accordance with all legal, ethical and institutional requirements and was approved by the UK Home office (PPL 7009066).

Treg isolation
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll density gradient centrifugation.CD4 + CD25 + T cells were isolated by negative selection of CD4 + T cells followed by positive selection of CD25 + T cells using the human CD4 + CD25 + T Regulatory Cell Isolation Kit (130-091-301, Miltenyi Biotec).The purity of the isolated cell product was between 90 and 98%.Aliquots of the HC CD4 + CD25 - effector T cells (Teff) were cryopreserved and used as responder cells in subsequent suppression assays, as described below.
For some experiments CD4 + CD25 + CD127 lo/-Tregs were isolated using flow cytometry-based sorting.In brief, CD4 + T cells were enriched by negative selection from PBMCs, as described above, and subsequently stained with anti-CD4, anti-CD25 and anti-CD127 antibodies.

Tregs
were sorted based on CD4 + CD25 + CD127 lo/-on a three laser FACS Aria high speed cell sorter (BD Biosciences).
To isolate healthy CD4 + CD25 + Tregs for culture in HC or LC serum, PBMCs from healthy donors was obtained from anonymized human leukocytes cones, supplied by the National Blood Transfusion Service (NHS Blood and Transplantation, Tooting, London, UK).CD4 + T cells were enriched using Rosette-Sep Human CD4 + T cell enrichment cocktail (15062, Stem Cell Technologies) according to the manufacturer's protocol and isolated by Ficoll density gradient centrifugation.CD25 + T cells were then labelled and separated from CD4+ T cells by positive magnetic separation using Human CD25 Microbeads II and LS columns (130-092-983, Miltenyi Biotec).

Flow cytometry phenotyping
Flow cytometry samples were acquired on a BD LSRFortessa flow cytometer (BD Biosciences) and analysed using the latest FlowJo software.For the staining, we first labelled dead cells with the Live/Dead Fixable dead cell stain kit (Violet L34964, Green L34970, Near-IR Red L34975; Invitrogen) in Phosphate-Buffered Saline (PBS, Gibco) for 10 min on ice.Staining of extracellular markers was performed in Staining Buffer (PBS + 0.9% Bovine Serum Albumin (BSA; Sigma Aldrich)) for 30 min on ice.In case of intracellular antigen labelling, the cells were fixed and permeabilized using the eBioscience Fixation/Permeabilization reagents (Fixation/Permeabilization Concentrate 00-5123-43, Fixation/Permeabilization Diluent 00-5223-56; ThermoFisher) for 30min at RT.
ROS was investigated by incubating the cells with 10 μM dihydroethidium (DHE; D11347, Invitrogen; 35 ) for 30 min at 37 • C followed by a washing step in Hanks' balanced salt solution (HBSS) without phenol red supplemented with diethylenetriamine-pentacetic acid (DTPA, 100 μM).The fluorescent probe BODIPY 581/591 1 μg/ml (D3861, Invitrogen) was used as an indicative of lipid peroxidation by LC or HC Tregs according to previous published reports. 36The levels of cell surface thiol groups were determined by 15 min incubation of CD4 + CD25 + cells with Alexa 633maleimide (10 μM; A20342; Invitrogen) at 4 • C as previous described. 37A complete list of all commercially available fluorochrome-conjugated anti-human and antimouse monoclonal antibodies used in the study are summarised in Table S4.

Cytokine bead array
To measure the cytokine concentration in HC and LC patient serum we employed the LEGENDplex Multi-Analyte Flow Assay kits (Human Cytokine panel 2 and Human Th Cytokine panel) according to the manufacturer's instructions.

Electron microscopy imaging
For mitochondrial imaging, 0.5 × 10 6 Treg cells were fixed in 2.5% glutaraldehyde in 100 mM sodium cocodylate, washed in cocodylate buffer.After dehydration samples were embedded in Eponate 12 resin (Ted Pella) and sections were cut.Images were acquired using a FEI Tecnai 12 Transmission electron microscope equipped with a TIETZ digital camera.Cristae width was measured using ImageJ software and averaged over 50 independent images, acquisition of EM micrographs and measurements of max cristae width displayed were performed using ImageJ software (NIH).Brightness and contrast were adjusted in ImageJ software (NIH).

RNA sequencing
RNA was extracted from freshly isolated CD4 + CD25 + CD127 lo/-Tregs of HC and LC using the ZymoResearch Direct-zol DNA/RNA MiniPrep (R2080, ZymoResearch) kit according to the manufacturer's protocol and Standard RNA-Sequencing was performed by Genewiz (Azenta Life Sciences).Raw reads were analysed for data quality using FastQC v0.11.5 (Babraham Bioinformatics) and filtered using skewer v0.2.2 38 for removing the low-quality reads and trimming the Illumina adapter.STAR program 39 against Human genome (GRCh38) was used for mapping the reads followed by the quantification of genes with the RSEM program 40 using GENCODE v26 reference annotation. 41fter eliminating genes without an expected value greater than 10, we carried out DESeq2, 42 a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability.For functional pathway analyses, we applied both Gene Set Enrichment Analysis (GSEA) from the clusterProfiler R package and Single-Sample Gene Set Enrichment Analysis (ssGSEA) from the GSVA R package, 43 using the curated gene set collection of canonical pathways (C2) and Gene Ontology (C5) available from the Molecular Signatures Database (MSigDB).Gene sets with <15 or >500 genes were excluded from the analysis.Among the top 15 pathways most significantly over-or under-represented when comparing LC and HC (Table S2), those associated with immunometabolism and redox homeostasis are shown (GSEA enrichment plots) in Figs.2j, k and 3f.

Nuclear Nrf2 translocation by ImageStream
Levels of nuclear Nrf2 were evaluated in freshly isolated CD4 + CD25 + Tregs (500-5 ′ 000 cells per group) following 12 h stimulation with prostaglandin J2 (PGJ2) using imaging flow cytometry.Nuclei were stained with DAPI.Cell images were acquired using an ImageStreamX MKII (Merck Millipore) and analysed with the IDEAS 6.2 software (NIH).The degree of nuclear translocation was assessed by quantifying the peak pixel intensity values for NRF2 staining within the digitally masked nuclear region (DAPI) in each individual sample.

Preparation of cell lysates for ELISA
Snap frozen cell pellets of freshly isolated Tregs of HC and LC patients were thawed on ice.RIPA Cell Lysis Buffer 2 (Enzo Life Sciences, ADI-80-1284) containing a protease inhibitor cocktail (1:200 dilution, Sigma-Aldrich, P1860-1 ML) was added at a concentration of 4 × 10 6 cells/ml.The cell pellet was resuspended by gentle pipetting and homogenised by passing through a 25G needle (BD Medical, 305125).The suspension was incubated on ice for 30 min and mixed intermittently.Following this the suspension was centrifuge at 21,000 g for 10 min in a 4 • C refrigerated microfuge.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)
The expression pattern of 5 Nrf2 targets genes (NQO1, GCLM, SOD1, GPX2, GSTA1) and 1 housekeeping gene (ACTB) was measured in freshly isolated peripheral blood CD4 + CD25 + Tregs of HC and LC individuals by qPCR using the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems).Total RNA was extracted using the Quick-RNA MiniPrep kit (R1054, ZymoResearch) according to the manufacturer's protocol and reverse transcription was performed using the Reverse transcription High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Cat.4374966).qPCR reactions were run with TaqMan Universal MasterMix II, no UNG (Applied Biosystems, Cat.4440040) and TaqMan Gene Expression Assay Probes (Applied Biosystems; ACTB Hs01060665_g1 (VIC), NQO1 Hs01045993_g1 (FAM), GCLM Hs00978072_m1 (FAM), SOD1 Hs00533490_m1 (FAM), GPX2 Hs01591589_m1 (FAM) GSTA2 Hs00747232_mH (FAM)).To quantify the mRNA transcript levels, the expression of target genes was normalized to the housekeeping gene ACTB and the results were presented as relative expression between cDNA of the target samples and a calibrator sample (independent healthy donor) according to the comparative ΔCT method.All qPCR experiments were performed in duplicates.

Serum culture experiments
Freshly isolated CD4 + CD25 + Tregs of healthy anonymized donors were cultured in X-vivo 15 supplemented with 25% serum of HC or LC patients for 24 h at 37 • C, 5% CO 2 .The cultured cells were then subject to flow cytometry, in vitro suppression assays or bioenergetic analysis as described above.In the culture conditions of Seahorse experiments anti-CD3/CD28 Dynabeads were used at a cell-to-bead ratio = 5:1.

Statistics
Statistical analysis was carried out in GraphPad Prism 9 (Version 9.5.1 GraphPad software Inc.) and IBM SPSS Statistics (version 28.0.1.1).Normally distributed data were summarised as mean and standard deviation (SD) and non-normally distributed data as median and interquartile range (IQR).Pairwise comparisons for normally distributed data were performed with unpaired t-tests, applying Welch's correction when F-tests indicated significant difference in variances.In cases in which data was not normally distributed, we log10 transformed the values to mitigate the skewness and performed a parametric unpaired t-test analysis.We performed a non-parametric Mann-Whitney test only when log10 transformation did not improve the distribution, because non-parametric tests have less power than parametric tests when sample sizes are small.To compare three or more groups that are defined by only one factor an ordinary one-way ANOVA was applied.When comparing groups with repeated measurements, we used linear mixed-effects models, to account for the relatedness between samples from the same individual.To examine whether differences between patients and controls are influenced by a given factor, we used repeated measures two-way ANOVA, or linear mixedeffects models with an interaction term between disease status and factor, following log-transformation for skewed distributions.To account for multiple comparisons, we applied Bonferroni correction and derived adjusted p-values.We calculated correlations using Pearson's correlation coefficient.A two-tailed p-value <0.05 (following Bonferroni's correction when required) was considered as statistically significant.The statistical tests performed for each experiment are indicated in the figure legends, and a per-graph summary of the statistical analyses is outlined in Table S3.The values are either shown as mean (SD) for approximately normally distributed data or median (IQR) for non-normally distributed data.For ease of interpretation, data sets that were log10 transformed are shown as median (IQR) in Figures S1 and S2 and Table S3.

Role of funding source
The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Circulating Tregs from LC patients are reduced in number and show phenotypic instability and functional impairment
To investigate the phenotype of circulating Tregs in patients with clinically stable LC, we recruited 108 patients together with 71 healthy controls (HC).The majority of patients were male, and the median Model for End-stage Liver Disease (MELD) score, as measure of liver disease severity, was 12 (Table 1).The proportion of circulating CD4 + CD25 + CD127 lo/-Tregs was significantly decreased in LC patients as compared to HCs (p = 0.014; Fig. 1a and b, Fig. S1a) despite comparable numbers of CD3 + CD4 + cells (p = 0.88; Fig. S1b).Of note, while HC and LC patients were not perfectly matched in what regards age and sex (Table 1), we observed no statistically significant correlation between these 2 parameters and the number of circulating CD4 + CD25 + CD127 lo/-Tregs (data not shown).The lower Treg fraction noted in LC patients was associated with a significant increase in the frequency of Annexin V + cells, indicative of a higher rate of Treg apoptosis (p = 0.011; Fig. 1c).Flow cytometric analyses revealed that, in comparison to HCs, LC patients displayed a significantly lower percentage of Tregs expressing the transcription factor Helios (p = 0.0022) and CTLA-4 (p = 0.0097; Fig. 1d and Fig. S1c).No statistically significant difference was seen in the percentage of FoxP3 + , CD39 + or HLA-DR + cells.[48] We next investigated whether these phenotypic changes observed in LC Tregs, which are suggestive of lineage instability, were associated with functional abnormalities. 49,50First, we exposed HC and LC Tregs to pro-inflammatory cytokine mixtures.This resulted in a significant increase in the expression of IL-17, but not IFNγ by LC Tregs as compared to HCs (IL-17 Mix 1: p = 0.0055, Mix 2: p = 0.0015, IFNγ Mix 1: p = 0.044, Mix: 2 p > 0.99 Fig. 1e).Of note, at baseline there was a trend towards higher IL-17 and IFNγ expression by LC Tregs (IL-17 p = 0.11, IFNγ p = 0.28; Fig. S1f).Secondly, we determined the capacity of freshly isolated Tregs from LC and HC patients to suppress the proliferation of CFSE-labelled CD4 + CD25 − third-party effector T cells.LC Tregs harboured a defective suppressive capacity as compared to HCs (Fig. 1f).Importantly, circulating Tregs isolated from patients with LC secondary to non-alcoholic steatohepatitis (NASH) with no prior alcohol history exhibited a degree of Treg functional deficit that was similar to Tregs from patients with alcohol-related LC (ARC), indicating that Treg impairment in LC is irrespective of the aetiology of liver disease (Fig. 1g).The magnitude of Treg dysfunction was inversely correlated with the severity of LC, as assessed by the MELD score (r = −0.82,p < 0.0001; Fig. 1h).Of note, no significant correlation was observed between Treg function and each individual constituent of the MELD score (serum creatinine, INR, bilirubin) (Fig. S1g).The phenotypic and functional abnormalities noted in the Tregs from LC patients were not associated with marked changes in the serum levels of circulating pro-inflammatory cytokines, which were found to be similar in HC and LC patients (Fig. S1h).In addition, the differences between LC and HC Tregs did not reflect changes in the epigenetic imprinting at the Treg-specific demethylated region (TSDR) of the FoxP3 locus (Fig. 1i).

Circulating Tregs from LC patients exhibit increased intracellular ROS and changes in mitochondria morphology
0][31] Furthermore, experiments conducted in animal models indicate that oxidative stress can induce Treg apoptosis. 51,52Based on these studies, we sought to determine whether LC influences the redox homeostasis of Tregs, which may in turn provide an explanation for their increased apoptosis (Fig. 1c).As compared to HCs, Tregs from LC patients exhibited increased intracellular ROS levels (p = 0.0090; Fig. 2a), lipid peroxidation (p = 0.013; Fig. 2b), as well as a reduction in cell surface free thiol groups (p = 0.15; Fig. 2c), altogether supportive of a state of increased oxidative stress.4][55] We assessed the activation status of the pro-oxidant Nox2 by analysing the phosphorylation and co-localization of its subunits in the cell membrane. 56Confocal microscopy analysis of freshly isolated Tregs revealed that in LC the cytosolic factor p47 phox co-localized with the gp91 phox Nox2 subunit at the cell membrane considerably more than in HC Tregs, suggestive of a higher Nox2associated ROS production (p = 0.049; Fig. 2d).In addition, levels of mitochondrial ROS were significantly higher in LC Tregs as compared to HC Tregs (p < 0.0001; Fig. 2e).To assess mitochondrial function of LC and HC Tregs, we performed metabolic flux analysis using the Seahorse Analyser (Fig. 2f).As compared to HC, LC Tregs had compromised mitochondrial respiration, with significantly reduced basal and maximal OCR (p = 0.028 and p = 0.058, respectively), as well as impaired ATP Production (p = 0.0008).These changes were paralleled by an increase in basal glycolysis (p = 0.12), decrease in glycolytic capacity (max.ECAR; p = 0.025) and a decrease in basal OCR/ECAR ratio (p = 0.0018; Fig. 2g).We also evaluated mitochondrial integrity by electron microscopy.][59] To further elucidate the molecular pathways underpinning these changes, we compared the transcriptional profile of freshly isolated HC and LC Tregs.Overall, the 2 cell populations differed in 267 differentially expressed genes (p < 0.01).Among the top 15 differentially regulated transcripts, we identified the gene encoding for the pro-apoptotic protein Bcl-2-modifying factor (BMF), a scaffolding protein involved in mitochondrial cristae integrity (CHCHD3), and the regulator of the mitochondrial redox sensor calcium-uniporter (MCUB) (Fig. 2i and Table S1).Gene set enrichment analysis (GSEA) revealed that LC Tregs exhibited an under-representation of gene sets involved in fatty acid metabolism, mitochondrial fatty acid beta oxidation and the pentose phosphate pathway (Fig. 2j and Table S2).1][62][63] Furthermore, LC Tregs showed enrichment in immune activation pathways such as IFNG, and under-representation of ribosome biogenesis pathways (Fig. 2k), which constitutes a hallmark of cell senescence and has been recently reported to be associated with decreased Treg proliferative capacity in vitro. 64Of note, Tregs expressing IFNγ and exhibiting reduced suppressive capacity are found in patients with autoimmune diseases such as multiple sclerosis. 65e Nrf2/HO-1 redox pathway is dysregulated in Tregs from LC patients The balance between ROS production and antioxidant signalling maintains a cell's redox homeostasis.In view of this, we next investigated whether alterations in the Nrf2 anti-oxidant signalling pathway could be an additional driver of the redox imbalance observed in LC Tregs.Whereas Nrf2 expression level was comparable in LC and HC Tregs (p = 0.59; Fig. 3a), Nrf2 nuclear translocation in response to Prostaglandin J2 (PGJ2) stimulation was significantly impaired in LC patients (HC p = 0.0009; LC p > 0.99; Fig. 3b).In line with this finding, LC Tregs exhibited substantially lower levels of HO-1 (p = 0.019; Fig. 3c and Fig. S2a), a trend towards higher levels of the HO-1 substrate Heme (Fig. S2b), and no significant changes in the protein expression of Bach1, which acts as a negative regulator of HO-1 (p = 0.71; Fig. 3d).To confirm the reduced Nrf2 activity, we quantified the transcript levels of two well-characterised targets of Nrf2, NQO1 and GCLM, which were found to be lower in LC than in HC Tregs (NQO1 p = 0.0012, GCLM p = 0.0028).SOD1 did not differ between the two groups (p > 0.99), whereas no expression was detected for GSTA2 and GPX2 in our study cohort (Fig. 3e).The existence of a dysregulated Nrf2 pathway was further supported by the under-representation in the LC Treg transcriptome of the "Response to Oxygen radical" pathway, which contained key Nrf2 downstream targets among its core enrichment genes (NQO1, CD36; Fig. 3f). 14,66o determine the extent to which Nrf2 impacts on Treg phenotype and function, we evaluated the characteristics of Tregs isolated from mice with a germline deficiency of Nrf2 (Nrf2 −/− ).Tregs from Nrf2 −/− mice exhibited decreased protein expression of FoxP3 (p = 0.0042), CD39 (p = 0.033) and CTLA-4 (p = 0.039, Fig. 3g).Furthermore, in keeping with our observations in LC patients, Nrf2 −/− Tregs displayed an increased rate of apoptosis (p = 0.0007; Fig. 3h) and reduced suppressive function (Fig. 3i).
(WT) or when Tregs were electroporated with a CRISPR/Cas9 complex not targeting the human genome (NC).HO-1 knock-out efficiency was almost 80% (Fig. 3j), which was confirmed by assessing the proportion of inserts and deletions (Indels) induced by CRISPR/Cas9 in the HMOX1 (HO-1) locus (Fig. S3).Knock-out of HO-1 did not influence the suppressive capacity of Tregs (Fig. 3k).In contrast, we observed that HO-1 was involved in Treg survival, given that, as compared to control cells, HO-1 knock-out Tregs were more prone to cell death when exposed to an environment of oxidative stress (Fig. 3l).
Taken together, these results indicate that an impaired Nrf2 pathway has a negative impact on the phenotype, function, and viability of Tregs, with the Nrf2 downstream target HO-1 being responsible for promoting Treg survival.

Exposure to LC serum recapitulates some of the Treg functional abnormalities observed in LC patients
8][69] We therefore sought to replicate the environment found in the circulation of LC patients by culturing healthy Tregs in LC patient serum for 24 h.Although culture in LC serum did not modify FoxP3 expression levels (p = 0.89, Fig. 4a), it did significantly increase the number of apoptotic Tregs (p = 0.015; Fig. 4b).In addition, LC serum induced a higher proportion of IL-17-, but not IFNγ-producing Tregs, which is indicative of Treg instability (IL-17 p = 0.025; IFNγ p = 0.37; Fig. 4c).The suppressive capacity of Tregs cultured with patient serum was reduced and mirrored the impaired function observed in LC Tregs (Fig. 4d).We also performed metabolic phenotyping of HC Tregs, using the Seahorse Analyser following a 24-h culture with LC or HC serum (Fig. 4e).As compared to HC serum, culture in the presence of LC sera increased basal glycolysis (p = 0.013) and the glycolytic capacity of HC Tregs (p = 0.016; Fig. 4e and  f), with no significant changes in mitochondrial respiration being observed.Taken together, although culture of HC Tregs in LC serum for 24 h did not completely mirror the aberrant Treg phenotype and metabolic changes observed in LC, it recapitulated the reduced suppressive capacity of LC Tregs, which could be directly attributed to their enhanced glycolytic activity. 62,70,71

Discussion
Treg-orchestrated anti-inflammatory feedback loops are indispensable to maintain self-tolerance and immune homeostasis.Oxidative stress, via oxidation of membrane lipids and proteins, regulates cell death and contributes to the pathogenesis of all types of inflammatory disorders.The impact of redox balance on Tregs, however, has not been thoroughly elucidated and remains contentious.Thus, depending on the in vitro experimental conditions employed, Tregs have been shown to be either more resistant 72 or conversely more vulnerable 67 than other lymphocyte subsets to the cytotoxic effects of free oxygen species.We previously reported in mice that Nox2 deficiency enhances Treg suppression by promoting the nuclear translocation of FOXP3 and p65/NF-KappaB and increasing CTLA-4, CD39, CD73 and GITR protein levels. 12Given the relevance of activation of Nox2 as a source of ROS, these data provided an indirect proof of the harmful effects of oxidative stress on Treg phenotype and function.Our current study reveals that in patients with cirrhosis and liver failure, circulating Tregs exhibit increased oxidative mediated damage, evidenced by higher intracellular levels of ROS and lipid peroxidation with loss of mitochondrial integrity, which compromises their viability and functional properties.
Tregs rely on lipid metabolism and fatty acid oxidation for their survival and function. 61,73In this regard, a previous study identified oxidative-mediated mitochondrial damage as a mechanism of Treg dysfunction in patients with autoimmunity. 68Our data linking impaired redox homeostasis, mitochondrial damage, and compromised fatty acid beta oxidation, extend the observations originally made in autoimmunity to patients with LC.Furthermore, our findings provide a mechanistic explanation to account for these findings based on the imbalance between the two key nonmitochondrial regulators of intra-cellular redox homeostasis, namely the anti-oxidant Nrf2 pathway and the ROS-producing Nox2 complex.We previously described in cardiac cells that the activation of Nrf2 by NADPH oxidases is a key feed-back loop to prevent ROS build-up and oxidative-mediated cell damage. 74Additional experiments in glioneuronal cells showed that Nrf2 deficiency results in increased Nox2 activity and ROS levels. 75Our current findings indicate that in LC Tregs, the cross-talk between Nox2 and Nrf2 is severely disrupted due to reduced Nrf2 nuclear translocation upon stress.Of note, a dysregulated Nrf2 signalling can directly affect mitochondrial morphology and integrity. 76,77Furthermore, a defective Nrf2 system can disrupt both fatty acid oxidation 78 and the pentose phosphate pathway. 63These published evidences, which are in keeping with the results of our electron microscopy and RNASeq experiments, respectively, further support the role of an impaired Nrf2 pathway as the key driver of the Treg abnormalities observed in LC.
In addition to the metabolic dysregulation and mitochondrial dysfunction outlined above, our study has identified an additional mechanism contributing to the reduced viability of Tregs in LC, which involves HO-1.][81] By conducting gene deletion experiments we now demonstrate that HO-1 is a key determinant of human Treg survival when exposed to oxidative stress.
From the liver disease standpoint, our study reveals that an impaired Treg function is a yet unexplored feature of cirrhosis-associated immune dysfunction (CAID).To control for potential clinical confounders and unambiguously clarify the involvement of Tregs in CAID, we chose to study a well-defined cohort of cirrhotic patients at risk of clinical decompensations due to the presence of portal hypertension, but who were clinically stable at the time of analysis.The fact that the Treg deficit was detected even in patients who were clinically stable, had no signs of acute decompensation, and did not yet exhibit markers of systemic inflammation in the circulation, is an important aspect of our study that contrasts with most reports published to date on the pathogenesis of CAID, which have focused on very advanced forms of chronic liver disease (i.e.patients with acute decompensation and/or multi-organ failure). 82In view of the magnitude of the Treg functional deficit described here, and given the central role of this immune cell subset in preventing immune mediated disorders, we speculate that Treg dysfunction may predispose cirrhotic patients to develop systemic inflammation and immunopathology, by failing to counteract the effector function of key pro-inflammatory players such as cytotoxic T cells, monocytes and dendritic cells.
The Treg abnormalities described in our report are strikingly similar to what has been observed in paradigmatic autoimmune diseases such as type-1 diabetes, multiple sclerosis, and rheumatoid arthritis. 7A fundamental question that remains unanswered in autoimmunity is which are the mediators responsible for Treg dysfunction.Potential explanations include both cell intrinsic mechanisms linked to the patients' genetic susceptibility to autoimmunity and the effect of systemic inflammatory cues.In view of the result of culturing healthy Tregs with LC serum, cell extrinsic mediators influencing the Treg's redox balance machinery are much more likely to explain our findings than cell intrinsic mechanisms.The former could be related to the inflammatory microenvironment, metabolomic landscape, gut microbiome and/or the patients' nutritional status.It is of importance to acknowledge that the study presented here has limitations derived from the use of relatively small sample sizes in some of the downstream experiments.As a result, our work cannot fully capture the clinical heterogeneity of the full spectrum of patients with chronic liver diseases.Future work conducted on larger patient cohorts will be required to account for therapy status, disease aetiology and stage and other confounders such as age and sex.
To conclude, by studying circulating Tregs from patients with stable cirrhosis we have uncovered a previously unrecognised immunoregulatory deficit that likely precedes the innate immune abnormalities that characterise CAID.Furthermore, we have gained insight into how the Nrf2-Nox2 signalling pathways and redox homeostasis influence Treg function and longevity.Our findings provide a strong rationale for exploring the therapeutic effects of Nrf2 activators and/or antioxidants in an attempt to modulate the inflammatory cascade at early stages of cirrhosis and potentially reduce the incidence of disease progression and clinical decompensations.

Data sharing statement
Datasets are available from the corresponding author on reasonable request.The dataset generated by RNA-Sequencing is available on the Gene Expression Omnibus (GEO) public database (GEO identifier GSE234857).

Declaration of interests
TV is currently employed by Janssen-Cilag Ltd (a subsidiary of Johnson & Johnson) and owns Johnson & Johnson stock/stock options.GL, ASF and MML are Founders of Quell Therapeutics Ltd.MR and MML are employed by Quell Therapeutics Ltd.PN received funding from Novo Nordisk; is advisory board member and received consulting fees from Novo Nordisk, Boehringer Ingelheim, Gilead, Intercept, Poxel Pharmaceuticals, BMS, Pfizer, Sun Pharma, Madrigal, GSK; speakers fees from Novo Nordisk, AiCME; and travel support from Novo Nordisk.The authors declare that the research in this manuscript was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Contributors
Study conceptualisation: NS, GL, ASF.Funding acquisition: NS.Data acquisition, analysis, and interpretation: EL, TV, NS, DMC, MR, SC, QP, EC, MC, JL, EPe, AZ, JE-H.Patient and Healthy control recruitment: TV, NS, LS, NSh.Reagent and material support: NL, RK.Verification of underlying data, figure preparation and writing of manuscript: NS, EL, TV, ASF.Critical revision of paper and important intellectual content: NS, EP, MT, MML, PN, AS, RIL, ASF, GL.All authors have read and approved the final version of the manuscript.

Table 1 :
• C followed by the intracellular staining for IL-17 and IFNγ.Demographic and clinical characteristics of study participants.
• C and 5% CO 2 proliferation of Teffs was determined as dilution of the CFSE dye.The suppressive ability of Tregs was assessed as the relative proliferation of Teffs in the presence of Tregs in comparison to the proliferation of Teffs alone.